In the last year we have begun to study purified PN to establish its physiological functions and possible relationship to cancer. Studies of its kinetics of inhibition by various serine proteases demonstrate that PN is a broad specificity inhibitor of arginyl-\or lysyl-specific serine proteases. Thus, PN inhibits bovine trypsin with an association rate constant of 2 X 106 M-1 S-1, thrombin (4 x 105 M-1 S-1), urokinase (2 x 105 M-1 S-1), plasmin (2 x 105 M-1 S-1), two chain tissue-type plasminogen activator (1 x 104 M-1 S-1), Factor Xa (3 x 103 M-1 S-1), NGF gamma subunit (2 x 103 M-1 S-1), and single chain tissue-type plasminogen activator (2 x 103 M-1 S-1). These results indicate that PN inhibits a variety of plasminogen activators and other fibrinolytic proteases. The breadth of specificity of PN suggests that the nature of the key physiologic target proteases may be determined by propinquity. We are currently examining the interaction of fibroblast PN with tryptase, a lys/arg-specific serine protease that is present in human dermis. Studies on the mechanism of regulation of plasminogen activator by human fibrosarcoma (HT1080) cells are nearing completion. These cells have been found to be like normal human fibroblasts in that they release plasminogen activator only in a latent form (urokinase proenzyme) and also release PN. However, these cells differ from normal fibroblasts in that they release vastly elevated amounts of the urokinase proenzyme and depressed amounts of PN. When HT1080 cells are grown on extracellular matrix (ECM) produced by smooth muscle cells they, unlike normal fibroblasts, degrade the matrix at a rapid rate. The ECM degradation is inhibited by about 50% by anti-urokinase antibody and is plasminogen-dependent, indicating that it involves cell plasminogen activator activity. Purified PN completely inhibits the ECM degradation for several days. However, thereafter the cells degrade the ECM at a rapid rate that is completely insensitive to PN. This suggests that the fibrosarcoma cells switch from one mechanism of degradation to another. The types of proteolytic activities involved in both mechanisms will be investigated. (B)